Over the years, many cytotoxic compounds have been discovered which are of potential use in cancer chemotherapy. Nitrogen mustards from one important family of such cytotoxic compounds. Nitrogen mustards are nitrogen analogs of mustard gas, S(CH2CH2Cl)2. The clinical use of cytotoxic compounds in general, and nitrogen mustards in particular, has been limited because of the poor selectivity in the cytotoxic effect between tumour cells and normal cells.
One approach to overcome this problem has involved the development of so-called prodrugs which are derivatives of the cytotoxic drug, often relatively simple derivatives, whose cytotoxic properties are considerably reduced compared to those of the parent drugs. Numerous proposals have been made for the administration of such prodrugs to patients under regimes whereby the prodrug is only converted to the cytotoxic drug in the region of the intended site of action, for example, by the action of an enzyme.
A variety of systems exist for delivery of the enzyme. One such system is described in WO 88/07378, and involves conjugating the enzyme to an antibody specific for a tumour marker, delivering the antibody enzyme conjugate to a patient, allowing the conjugate to localise, and then delivering the prodrug to the patient. This system is referred to as “antibody-directed enzyme prodrug therapy” (ADEPT).
Various other proposals for delivery of the enzyme to the desired site of action exist, including “gene-directed enzyme prodrug therapy” (GDEPT), in which a vector such as a viral vector carrying a gene encoding the prodrug-converting enzyme is delivered to cells at the desired site of action (Huber et al, Proc. Natl. Acad. Sci. USA, (1991) Vol. 88, p. 8039). GDEPT is also described in WO 96/03151.
A further alternative system, often referred to as “ligand-directed enzyme prodrug therapy” (LIDEPT), is to provide a ligand, generally a naturally occurring polypeptide whose biological role involves its binding to a cognate receptor on the surface of the cell, conjugated to the prodrug-activating enzyme. Such a system is described in WO 97/26918, where VEGF is particularly exemplified as an example of a ligand.
One class of prodrugs suggested for use in the above systems is that of prodrugs of nitrogen mustard compounds. Benzoic acid nitrogen mustards, such as the one shown below, are bifunctional alkylating agents, and a variety of prodrugs of such compounds are described in the art. 
One class of such prodrugs comprise a protecting group which may be removed by the action of a carboxypeptidase enzyme, such as bacterial carboxypeptidase G, including such enzymes available from Pseudomans species, such as carboxypeptidase G2 (CPG2). In many cases, the carboxylic acid group, —COOH, of the drug is protected as an amide, —CONHR, in the prodrug, for example, by reaction with an α-amino acid (e.g., glutamic acid) or a polypeptide chain, R—NH2. The prodrug is converted into the active drug by cleavage of the amide bond, —CONH—, between the residue of the α-amino acid or oligopeptide and the residue of the benzoic acid nitrogen mustard. This enzyme, and prodrugs which may be activated by it have been described in various publications. WO 88/07378 pertains to certain nitrogen mustard compounds of the following formula, where X and Y are —Cl and —OSO2CH3, or both —OSO2CH3, or both —OSO2CF3, and R is the residue of an α-amino acid RNH2, for example, glutamic acid. 
Springer et al., J. Medicinal Chem., (1990) Vol. 33, pp. 677-681 and Springer et al., Anti-Cancer Drug Design (1991), Vol. 6, pp. 467-479 pertain to certain nitrogen mustard compounds of the following formula, wherein X and Y are —Cl or —OSO2CH3. 
WO 94/02450 pertains to certain nitrogen mustard compounds of the following formula, wherein X is —O—, —NH—, or —CH2— and Y is =O. 
WO 94/25429 pertains to certain 2-fluoro- and 3-fluoro-substituted nitrogen mustard compounds of the following formula, wherein the group —NH—R is the residue of an α-amino acid NH2—R or oligopeptide NH2—R, and Y and L, which may be the same or different in a given molecule, are leaving groups: 
WO 96/03515 pertains to GDEPT systems which employ certain nitrogen mustard compounds of the following formula of the formulae shown below, in which X is —C(═O)—, —OC(═O)—, -NHC(═O)—, or —CH2—C(—O)— and Z1 is —O— or —NH—. 
WO 96/22277 pertains to certain nitrogen mustard compounds of the following Formula, in which Z1 is —O— or —NH—. 
Thus although a number of nitrogen mustard prodrugs exist in the art, there is a continuing need for improved prodrugs, which can provide enhanced efficacy, for example by having an improved ratio of toxicity between prodrug and active drug form, better half-life or more potency.